1. Technical Field
The present invention relates to the diagnosis of transmissible spongiform encephalopathies (TSEs). It especially teaches the production of a soluble prion protein (PrP)-chaperone complex and the advantageous use of such chaperone-PrP complex, especially in the detection of PrP in an immunoassay, as well as its use as an immunogen.
2. Background Information
Transmissible spongiform encephalopathies (TSEs) include kuru-kuru, Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker syndrome (GSS) and fatal familial insomnia (FFI) in humans, bovine spongiform encephalopathy (BSE), and scrapie in sheep. It is believed that the TSEs are caused by a novel class of infectious pathogens the so-called prion proteins or prions. According to the protein-only hypothesis the “infectious agent” is the prion protein in an abnormal oligomeric form which is called prion protein scrapie type (=PrPSc). The mammalian prion protein is highly conserved during evolution and normally is a monomeric cell surface glycoprotein which is termed PrPC, wherein C stands for cellular. Both isoforms share the same amino acid sequence but differ in their secondary and tertiary structure. It is believed, that only the PrPSc can transmit the disease, whereas PrPC appears not to be correlated with any disease mechanism leading to TSE.
Bovine spongiform encephalopathy (BSE) is a fatal, neuro degenerative, transmissible brain disease of cattle. The disease is fatal for cattle within weeks to months of its onset.
BSE first came to the attention of the scientific community in November 1986 with the appearance in cattle of a newly recognized form of neurological disease in the United Kingdom. Between November 1986 and November 2002, about 180 000 cases of BSE were confirmed in the United Kingdom.
Since 1989, when the first BSE case was reported outside the United Kingdom, relatively small numbers of cases (about 1500 in total) have also been reported in native cattle in many European countries and Japan. However, all but a couple of dozen have been reported in four countries—France, Ireland, Portugal and Switzerland.
Small numbers of cases have also been reported in Canada, the Falkland Islands, and Oman, but solely in animals imported from the United Kingdom.
Creutzfeldt-Jakob disease (CJD) is a rare and fatal neurodegenerative disease of unknown cause. Patients are usually aged between 50 and 75 and typical clinical features include a rapidly progressive dementia associated myoclonus and a characteristic electroencephalographic pattern. Neuropathological examination reveals cortical spongiform change, hence the term “spongiform encephalopathy”.
H. G. Creutzfeldt is credited with the first description of the disorder in 1920, although by current diagnostic criteria his case would be highly atypical. A year later another German neurologist, A. Jakob described four cases, at least two of whom had clinical features suggestive of the entity today recognized as CJD.
Although CJD appears to occur as a predominantly sporadic disorder it can also occur as a dominantly inherited or infective condition. The latter mode of transmission was first elucidated during the study of kuru-kuru in the 1950's. This neurodegenerative condition occurs only in the people of the Fore region of Papua New Guinea and is thought to have resulted from the consumption of brains during endocannabalistic funeral rituals. The similarities between kuru-kuru and scrapie, the transmissible spongiform encephalopathy of sheep, prompted a veterinary neuropathologist, Hadlow, to suggest that transmission studies of kuru-kuru be performed. The success of those studies and the recognition that the neuropathological changes in kuru-kuru were similar to those of CJD, was followed by the transmission of CJD to the chimpanzee by intracerebral inoculation of brain tissue. In 1974 a case of iatrogenic CJD due to corneal transplantation occurred and subsequently contaminated neuro-surgical instruments, dural grafts, and brain depth electrodes have all been recognized as transmitting the disease. In 1985 the first case was reported in a recipient of contaminated human derived growth hormone and subsequently over 60 similar cases have arisen world-wide in addition to 4 cases associated with human derived gonadotrophin. The familial occurrence of CJD has been recognized for many years but was unexplained. The discovery of linkage to a region on the short arm of chromosome 20 has led recently to the elucidation of various dominantly inherited mutations.
Variant Creutzfeldt-Jakob disease (vCJD) is a rare and fatal human neurodegenerative condition. Like Creutzfeldt-Jakob disease (CJD), vCJD is classified as a transmissible spongiform encephalopathy (TSE) because of characteristic spongy degeneration of the brain and the transmissibility of the disease. First described in March 1996, vCJD appears to be strongly linked to exposure to the BSE agent.
From October 1996 to early November 2000, 85 cases of vCJD have been reported in the United Kingdom, 3 in France and 1 in Ireland. Insufficient information is available at present to make any well-founded prediction about the future number.
The nature of the transmissible agent in CJD is the matter of some controversy. Previously considered a “slow virus” no viral agent has ever been convincingly demonstrated and no evidence of an immunological response seen. Additionally the infectious pathogen shows a remarkable resistance to treatments that would normally be expected to inactivate viruses. The viral hypothesis has been elegantly challenged by the prion (“proteinaceous infectious particle”) theory which states that the infectious agent is derived from a protease-sensitive protein (designated PrPC) which is a constituent of the normal cell membrane. It is postulated that the normal protein undergoes a post-translational conformational change forming the insoluble pathogenic form of the prion protein (PrPSSc). This in turn induces more of the normal PrPC to form PrPSc—hence a chain reaction is set in motion with the exponential production of the insoluble prion protein being formed. The initial abnormal prion protein needed to seed this process may occur spontaneously as a rare event (which would account for the low incidence of sporadic CJD), following inoculation (accounting for observed transmission phenomena) or when initiated by a genetic abnormality of the PrP gene. The mechanism by which PrPSc induces the pathological changes in CJD-spongiform change, gliosis, neuronal loss and (infrequently) plaques remains unclear. Although the prion hypothesis neatly explains many of the observed phenomena of transmissible spongiform encephalopathies (TSEs) it has one particular weakness. Scrapie is known to exhibit various “strains” characterized by different incubation periods, clinical features and pathology when transmitted. This is much more in keeping with a virus-like agent and strain variation, independent of the host genome, is difficult to reconcile with the prion theory.
The majority of CJD-cases are sporadic (85%), between 10-15% are familial and the remainder are iatrogenic.
CJD occurs worldwide with a roughly even incidence of between 0.5-1.0 cases per million per year. Higher rates (up to 100-fold) have been reported in Slovakia and Libyan-born Israelis but this is explained by the high incidence of a certain mutation of the PrP gene in these groups. The geographical distribution of CJD in the United Kingdom over the past 25 years demonstrates no overall evidence of spatiotemporal aggregation of cases, despite the occurrence of local areas of relatively high incidence over short periods. There is no evidence of case to case transmission and spouses of sporadic cases do not have an increased incidence of the disease.
TSEs are known to affect various animal species including sheep, goats, mink, mule deer, cows and recently cats. Scrapie, a disorder of sheep and goats, has been known for over 300 years and is endemic in the British Isles. In 1938 experimental transfer of scrapie from one sheep to another by inoculation provided evidence of an infective etiology. However there is no evidence of transmission of scrapie from sheep to man and there is no increased incidence of CJD in countries with scrapie compared to those without (e.g. UK and Australia).
The close relationship in pathogenesis between BSE and CJD has in recent years triggered massive scientific and industrial research into further understanding disease transmission pathogenesis as well as into early diagnosis of the underlying disease.
Research, however, is hampered by the fact that both PrPC, as well as PrPSc are difficult to isolate, to purify and especially to handle. PrP is a fairly sticky protein. Moreover, PrP is a metastable protein with a pronounced tendency to aggregate or precipitate in physiological buffers. Aggregation and precipitation of PrP is frequently observed under long term storage conditions and can be induced or accelerated by increasing storage temperature.
What render PrP even more critical is the biohazards associated with handling a PrP from a biological source, especially with regard to disease transmission.
Various attempts to obtain a prion protein by recombinant expression are known from the literature. Hornemann, S., et al., FEBS Letters 413 (1997) 277-281, for example describe the expression of full-length murine prion protein (mPrP (23-231)). These researchers expressed the complete amino acid sequence of the prion protein, mPrP (23-231), in the cytoplasm of Escherichia coli. The corresponding protein has been obtained in the form of inclusion bodies, which had to be solubilized by 8 M urea and oxidatively refolded. The human prion protein has been expressed in Escherichia coli by Zahn, R., et al., FEBS Letters 417 (1997) 400-404. In this paper the human prion protein (hPrP) from amino acid 23 to amino acid 231 has been expressed, as well as several fragments of hPrP. These investigators used polypeptides comprising a histidine tail fusion protein. This did facilitate the purification of the proteins which also had been obtained in the form of inclusion bodies. When purified, isolated and stored in a rather concentrated solution the hPrP thus obtained has been found to be stable.
However, if used as a standard material for immunoassays, hPrP, has to be diluted to comparatively low concentrations and nonetheless the diluted material has to be stable for several months.
Physiological, buffered salt solutions that mimic the extracellular fluids of human tissues have uniformly failed to solubilize PrP in solution if stored for weeks or months at 4° C.
Immunoassays in general are performed at physiological pH. Hence polypeptides soluble at physiological buffer conditions are extensively used in various immunoassay methods, such as ELISA (enzyme-linked immunosorbent assay), for example in the diagnosis of and screening for a certain disease.
Due to its aggregation tendency under physiological buffer conditions, and due to its metastable nature the PrP is difficult to use in an immunoassay E.g., in a sandwich assay format PrP is difficult to handle, because it tends to aggregate or even precipitate. Due to its metastable nature PrP may also lead to false results caused by unspecific binding.
In addition the metastability of PrP under physiological buffer conditions renders this protein a difficult target for routine (bio-) chemical procedures. The vast majority of “labeling chemistries”, i.e., the chemical procedures used for binding a label, e.g., a marker group to a polypeptide, are based on nucleophilic chemistry and thus rather restricted to a pH window from about pH 6 to about pH 8 and thus only work under more or less physiological buffer conditions. These routine procedures, e.g., as described in Aslam, M. and Dent, A., The preparation of protein-protein conjugates in “Bioconjugation”, eds. M. Aslam and A. Dent, McMillan Reference, London (1998), pp. 216-363, are difficult to carry out with PrP.
Therefore a tremendous need exists to provide PrP in a thermodynamically stable rather than in its metastable form. In this thermodynamically stable form PrP is for example soluble at physiological pH, stable in solution and/or easier to produce and/or handle.
There is also an urgent need to provide such PrP without encountering the biohazards associated with PrP that has been isolated from a mammal.